Surface sensor and preparation method and application thereof

Abstract

The invention belongs to the technical field of sensors, and particularly relates to a surface sensor and a preparation method and application thereof. The surface sensor comprises a sensing chip and a microfluidic channel, the sensing chip and the side, with the concave-convex surface, of the micro-fluidic channel are oppositely arranged to form a fluid channel; the micro-fluidic channel is provided with a groove structure; a convex structure is arranged on the bottom surface of the groove structure; micron columns are distributed on the bottom surface of the groove structure and the upper end surface of the convex structure; the protruding structures are perpendicular to the fluid direction and are arranged in an array mode. The convex structures and the micron columns are arranged in the micro-fluidic channel, so that a flow field in the micro-fluidic channel is changed, the flow speed of fluid is increased, the exchange rate of substances in the micro-channel is increased, the test error can be reduced, and the detection sensitivity is improved. When the surface sensor is used for detecting the AFP antigen, a secondary antibody is not required to be introduced, the response of 1ng / mL of AFP antigen can be detected, and the surface sensor has excellent sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of sensors, and in particular relates to a surface sensor and a preparation method and application thereof. Background Art

[0002] Plasmon resonance biosensors are increasingly being used in medical diagnosis, food safety, and environmental monitoring. Under the excitation of an external light source, the surface plasmon resonance (SPR) between the noble metal layer and the medium responds sensitively to changes in the refractive index (RI) of the surrounding medium. By measuring changes in these resonance conditions (e.g., angle, wavelength, intensity, or phase), molecular interactions occurring on the sensor surface can be directly monitored in real time. This unique property makes plasmon resonance biosensors an indispensable tool for real-time and label-free analysis. Surface refractive index sensors have great application potential due to their high sensitivity and versatility.

[0003] At present, in SPR sensing technology, the main ones are propagating surface plasmon (PSPR) and localized surface plasmon (LSPR). The propagation electric field length of PSPR is large, and the electric field of LSPR is generally localized within a few nanometers, with high sensitivity. Fiber optic surface plasmon resonance biosensor is mainly based on the fiber optic surface plasmon resonance sensing principle, and analyzes the characteristics of the measured biological molecules by detecting the change of refractive index in the evanescent field region of the fiber surface. In order to obtain high sensitivity, the above method needs to be coordinated with other detection methods (fluorescence, magnetism, electricity, etc.), and the analyte needs to be functionalized (modified label), such as modified fluorescent groups, magnetic groups, color-developing groups, etc. However, although PSPR has good sensitivity, it needs to rely on complex equipment and high costs, and it is difficult to transform from a large system and equipment into a small integrated instrument. The LSPR structure is relatively simple, but the line width of its resonance peak is relatively large and the quality factor is low, making it difficult to achieve industrial repeatability and reproducibility. Although fiber optic sensors have certain advantages in size, they have complex optical designs and high-precision processing and assembly, which are difficult to achieve in terms of production costs, processing difficulty and repeatability. The introduction of analyte modification labels adds additional steps, which correspondingly increases detection time, test errors and costs.

[0004] Therefore, it is of great significance to provide a surface sensor with high sensitivity, easy processing and low cost. Summary of the invention

[0005] The present invention aims to solve one or more technical problems existing in the above-mentioned prior art, and at least provide a beneficial choice or create conditions. Specifically, the present invention provides a surface sensor which has high sensitivity, is easy to process, has low cost, and can be converted into a small integrated instrument.

[0006] The inventive concept of the present invention is as follows: the surface sensor of the present invention comprises a sensor chip and a microfluidic channel; the sensor chip and the side of the microfluidic channel having a concave and convex surface are placed opposite to each other to form a fluid channel; the microfluidic channel has a groove structure; the bottom surface of the groove structure is provided with a convex structure; the bottom surface of the groove structure and the upper end surface of the convex structure are both distributed with micrometer columns; the convex structure is perpendicular to the fluid direction and arranged in an array. The provision of the convex structure and the micrometer column of the present invention changes the flow field in the microfluidic channel, and compared with the flow field without a structure, the flow velocity of the fluid increases, the material exchange rate in the microfluidic channel is accelerated, the influence of the Fabry-Perot (FP) interference phenomenon on the test spectrum is reduced, and the influence of the FP resonance is reduced or even eliminated, thereby reducing the test error and improving the detection sensitivity.

[0007] Accordingly, a first aspect of the present invention provides a surface sensor.

[0008] Specifically, the surface sensor comprises a microfluidic channel and a sensor chip; the sensor chip and the microfluidic channel are placed opposite to each other with the concave and convex surfaces thereof to form a fluid channel;

[0009] The microfluidic channel has a groove structure; the bottom surface of the groove structure is provided with a convex structure;

[0010] The bottom surface of the groove structure and the upper end surface of the protrusion structure are both distributed with micrometer columns;

[0011] The protrusion structures are perpendicular to the fluid direction and arranged in an array.

[0012] Specifically, the structural area of ​​the sensor chip and the structural area with concave and convex surfaces of the microfluidic channel are placed opposite to each other, and the non-structural area of ​​the sensor chip and the non-structural area of ​​the microfluidic channel are bonded together to form a fluid channel to obtain the surface sensor.

[0013] Preferably, the depth of the groove structure is 12-33 μm; the width of the groove structure is 200-1500 μm.

[0014] Further preferably, the depth of the groove structure is 10-30 μm; the width of the groove structure is 220-1000 μm.

[0015] More preferably, the depth of the groove structure is 10 μm; and the width of the groove structure is 500 μm.

[0016] Preferably, the groove structure is an I-shaped groove.

[0017] Preferably, the height of the protruding structure is 4.5-10 μm; the width of the protruding structure is 18-42 μm; the length of the protruding structure is 100-1000 μm; and the distance between adjacent protruding structures is 8-32 μm.

[0018] Further preferably, the height of the protruding structure is 5-10 μm; the width of the protruding structure is 20-40 μm; the length of the protruding structure is 100-1000 μm; and the distance between adjacent protruding structures is 10-30 μm.

[0019] More preferably, the height of the protruding structure is 5 μm; the width of the protruding structure is 30 μm; the length of the protruding structure is 500 μm; and the distance between adjacent protruding structures is 20 μm.

[0020] Preferably, the diameter of the micron column is 0.9-3.3 μm; the height of the micron column is 0.8-1.2 μm; further preferably, the diameter of the micron column is 1-3 μm; the height of the micron column is 0.9-1.1 μm.

[0021] Preferably, the micro-pillars are randomly distributed on the bottom surface of the groove structure and the upper end surface of the protrusion structure.

[0022] Specifically, compared with a flat microfluidic channel without a protrusion structure, the present invention changes the flow field in the microfluidic channel by setting a protrusion structure and micron columns of a certain size in the microfluidic channel, increases the flow rate of the fluid, accelerates the material exchange rate in the channel, reduces the influence of the FP interference effect on the test spectrum, reduces the test error, and improves the detection sensitivity.

[0023] Preferably, the microfluidic channel is provided with a hole at each end along the length direction, serving as an inlet and an outlet for the detection solution, respectively.

[0024] Preferably, the diameter of the hole is 0.3-1 mm; further preferably, the diameter of the hole is 0.65-0.75 mm; further preferably, the diameter of the hole is 0.7 mm.

[0025] Preferably, the sensor chip includes a substrate, a dielectric layer, and a metal layer in sequence, the dielectric layer has a plurality of dielectric grooves, and the plurality of dielectric grooves are periodically and evenly distributed; the opening width of the dielectric grooves in the direction from the groove mouth to the groove bottom gradually decreases; the metal layer is arranged on the dielectric layer and forms metal grooves corresponding to each of the dielectric grooves.

[0026] Specifically, the difference between the sensor chip of the present invention and the structure disclosed in the prior art with publication number CN113433094A is that the type of metal layer is different. The metal layer of the present invention includes at least one of a gold layer, a silver layer, an aluminum layer, and a copper layer, while the metal layer in the prior art is selected from at least one of a gold layer, a silver layer, and a platinum layer. The rest is the same as the prior art with publication number CN113433094A.

[0027] Preferably, in the sensor chip, the metal layer can be a single-layer structure of a metal layer, or a stacked structure composed of different metal layers; for example, the metal layer can be a single-layer structure of a gold layer, an aluminum layer, a silver layer or a copper layer, or a stacked structure composed of at least two of the gold layer, aluminum layer, silver layer and copper layer.

[0028] Specifically, gold has good chemical stability and biocompatibility and is a preferred material for biosensing. However, considering that gold is relatively expensive and that nanotransfer technology and the working environment of biosensor chips require a thicker metal layer (~300nm), the present invention preferably adopts a method of superimposing 50-100nm Au and 200-250nm other metal layers (Ag, Cu, Al or other low-priced, high-reflectivity metals) to reduce the processing cost of the biosensor while achieving the biosensing function, thereby facilitating the reduction of the cost of mass production.

[0029] The second aspect of the present invention provides a method for preparing the surface sensor according to the first aspect of the present invention.

[0030] Specifically, the preparation method of the surface sensor comprises the following steps:

[0031] The microfluidic channel and the side of the sensor chip having the concave and convex surface are placed opposite to each other to obtain the surface sensor.

[0032] Specifically, since the sizes of the structural features of the microfluidic channel and the sensor chip belong to different size classes, it is equivalent to placing the sides with structural features of the two in correspondence, and fitting the non-structural feature areas together. After coordination, the structural feature areas form a space that serves as a fluid channel for the detection fluid to pass through.

[0033] Preferably, the method for preparing the microfluidic channel comprises the following steps:

[0034] (1) forming randomly distributed micropores on the template to obtain a template having distributed micropores;

[0035] (2) covering the surface of the template obtained in step (1) with a mask material, forming a mask material having a grating structure by photolithography, and etching the template using the mask material having a grating structure as a mask to form an array-arranged grating structure on the template;

[0036] (3) covering the surface of the template obtained in step (2) with a mask material, forming a mask material covering the grating structure by photolithography, and etching the template using the mask material covering the grating structure as a mask to form auxiliary protrusions on the template;

[0037] (4) Covering the organic silicon mixture on the template obtained in step (3), curing and separating, and obtaining a microfluidic channel.

[0038] Specifically, in step (1), a mask material is covered on the surface of the template, and circular holes are obtained on the mask material by photolithography, and then etching is performed to form microholes at positions corresponding to the circular holes on the template.

[0039] Preferably, in step (1), the diameter of the micropore is 0.9-3.3 μm; the depth of the micropore is 0.8-1.2 μm; further preferably, in step (1), the diameter of the micropore is 1-3 μm; the depth of the micropore is 0.9-1.1 μm.

[0040] Specifically, according to the structural features of the designed microfluidic channel, the micropores are randomly distributed in the template area corresponding to the grating structure in step (2) and its vicinity, and it is ensured that the micropores are distributed at the grating structure position and between adjacent grating structures.

[0041] Specifically, in step (2), a mask material having a grating structure is used as a mask, so that the template position corresponding to the grating structure is not etched due to the protection of the mask material, while the material in other areas of the template is etched downward. After removing the mask material, an array-arranged grating structure is obtained on the template.

[0042] Specifically, in step (2), a grating structure is obtained on the template, the grating structure corresponds to the grooves between the convex structures of the final microfluidic channel, and the grooves between the grating structures correspond to the convex structures of the final microfluidic channel.

[0043] Preferably, in step (2), the height of the grating structure is 4.5-10 μm; the width of the grating structure is 8-32 μm; the length of the grating structure is 100-1000 μm; and the distance between adjacent grating structures is 18-42 μm.

[0044] Preferably, in step (2), the height of the grating structure is 5-10 μm; the width of the grating structure is 10-30 μm; the length of the grating structure is 100-1000 μm; and the distance between adjacent grating structures is 20-40 μm.

[0045] Preferably, in step (2), the height of the grating structure is 5 μm; the width of the grating structure is 20 μm; the length of the grating structure is 500 μm; and the distance between adjacent grating structures is 30 μm.

[0046] Specifically, in step (3), the mask material covers the grating structure due to the effect of photolithography. The grating structure is not affected by subsequent etching due to the protection of the mask material, and the template position corresponding to the mask material covering the grating structure will not be etched, while the material in other areas of the template will be etched downward. After removing the mask material, an auxiliary protrusion is obtained on the template, and the grating structure is located above the auxiliary protrusion.

[0047] Specifically, the auxiliary protrusion formed in step (3) corresponds to the groove structure of the microfluidic channel finally prepared.

[0048] Preferably, the mask material covering the grating structure is in an I-shaped shape, that is, the auxiliary protrusion obtained in step (3) is in an I-shaped shape, and the grating structure is located on the auxiliary protrusion structure in the I-shaped shape.

[0049] Preferably, in step (3), the height of the auxiliary protrusion is 12-33 μm; and the width of the auxiliary protrusion is 200-1500 μm.

[0050] Further preferably, in step (3), the height of the auxiliary protrusion is 10-30 μm; and the width of the auxiliary protrusion is 220-1000 μm.

[0051] Further preferably, in step (3), the height of the auxiliary protrusion is 10 μm; and the width of the auxiliary protrusion is 500 μm.

[0052] Preferably, before performing step (4), the template obtained in step (3) is cleaned with a cleaning agent to remove the mask material remaining on the surface of the template.

[0053] Preferably, the cleaning agent includes concentrated sulfuric acid and hydrogen peroxide.

[0054] Preferably, the mass fraction of the concentrated sulfuric acid is 95-98%, and the mass fraction of the hydrogen peroxide is 32-38%.

[0055] Further preferably, the mass fraction of the hydrogen peroxide is 35%.

[0056] Preferably, the volume ratio of the concentrated sulfuric acid to the hydrogen peroxide is (6-8):3; further preferably, the volume ratio of the concentrated sulfuric acid to the hydrogen peroxide is (6.5-7.5):3; further preferably, the volume ratio of the concentrated sulfuric acid to the hydrogen peroxide is 7:3.

[0057] Preferably, the cleaning agent is further rinsed with water, blown dry with nitrogen and dried after cleaning.

[0058] Preferably, in step (4), the organosilicon mixture comprises organosilicon and a curing agent.

[0059] Preferably, the mass ratio of the organosilicon to the curing agent is (8-12):1; further preferably, the mass ratio of the organosilicon to the curing agent is (9-11):1; further preferably, the mass ratio of the organosilicon to the curing agent is 10:1.

[0060] Preferably, the silicone comprises polydimethylsiloxane (PDMS).

[0061] Specifically, the silicone mixture adopts Dow Corning Sylgard 184.

[0062] Preferably, before curing, degassing is performed in a vacuum dryer.

[0063] Preferably, the curing temperature is 60-80°C, and the curing time is 1.5-2.5h; further preferably, the curing temperature is 65-75°C, and the curing time is 1.8-2.2h; further preferably, the curing temperature is 70°C, and the curing time is 2h.

[0064] Preferably, in step (4), in order to ensure smooth separation of PDMS from the template obtained in step (3), a release agent trichloro(1H, 1H, 2H, 2H-triacontafluorooctyl)silane (TFOCS) is evaporated on the template obtained in step (3). Specifically, the release agent is coated on the template obtained in step (3), and placed in a vacuum drying oven, evaporated, and taken out.

[0065] Preferably, the evaporation time is 25-35 min; further preferably, the evaporation time is 27-33 min; further preferably, the evaporation time is 30 min.

[0066] Specifically, step (4) is equivalent to a transfer process, in which a template corresponding to the structural features of a microfluidic channel is first prepared through steps (1) to (3), that is, a microfluidic channel template is obtained in step (3), and then the microfluidic channel of the present invention is obtained through a transfer process of step (4) (for example, the micropores of the microfluidic channel template become micrometer columns of the microfluidic channel after transfer, the auxiliary protrusions of the microfluidic channel template become groove structures of the microfluidic channel after transfer, the grating structure of the microfluidic channel template becomes grooves between protrusion structures of the microfluidic channel after transfer, and the grooves between the grating structures of the microfluidic channel template become protrusion structures of the microfluidic channel after transfer).

[0067] Preferably, after the transfer in step (4) is completed, a hole is provided at each end of the microfluidic channel along the length direction, serving as an inlet and an outlet for the detection solution, respectively.

[0068] Preferably, the diameter of the hole is 0.3-1 mm; further preferably, the diameter of the hole is 0.65-0.75 mm; further preferably, the diameter of the hole is 0.7 mm.

[0069] Preferably, the microfluidic channels are distributed in an array.

[0070] A third aspect of the present invention provides a surface sensor system.

[0071] Specifically, the surface sensor system includes the surface sensor described in the first aspect of the present invention.

[0072] Preferably, the surface sensor system further comprises a syringe pump, a light source generator, a semi-transparent and semi-reflective mirror, a microscope, a detector, and a data output window; the syringe pump is connected to the inlet of the microfluidic channel detection solution.

[0073] Preferably, the detector includes any one of a CCD photodetector, a CMOS photodetector, and a PIN photodetector.

[0074] The fourth aspect of the present invention provides a use of the surface sensor described in the first aspect of the present invention or the surface sensor system described in the third aspect of the present invention in detection and analysis for purposes other than disease diagnosis.

[0075] Preferably, the detection analysis includes antigen concentration detection.

[0076] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0077] (1) The surface sensor of the present invention comprises a microfluidic channel and a sensor chip; the microfluidic channel is a groove structure; the bottom surface of the groove structure is provided with micrometer columns and array-arranged protrusion structures; the upper end surface of the protrusion structure is distributed with micrometer columns. Compared with a flat microfluidic channel without a protrusion structure, the setting of the protrusion structure and micrometer columns of the present invention changes the flow field in the microfluidic channel. Compared with a flow field without a structure, the flow velocity of the fluid increases, the material exchange rate in the microchannel is accelerated, the influence of the FP interference phenomenon on the test spectrum is reduced, and the influence of FP resonance is reduced or even eliminated, thereby reducing the test error and improving the detection sensitivity. When the surface sensor of the present invention is used to detect AFP antigen, no secondary antibody is required. In the case of label-free (no marker signal amplification technology), a 1ng / mL alpha-fetoprotein (AFP) response (clinical 7ng / mL is the critical value) can be detected, and the S-type fitting is met in the range of 1-10000ng / mL.

[0078] (2) The metal layer of the sensor chip of the present invention has a smooth surface, which can effectively improve the smoothness of the metal layer, further reduce the resonance peak line width, and improve the quality factor. At the same time, it can effectively avoid the problems of metal layer thickness changes and metal layer asymmetry affecting the optical response of the biosensor. The present invention combines the microfluidic channel with the sensor chip, which can further reduce the test error and improve the detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 This is a schematic diagram of the structure of the microfluidic channel in Example 1 of the present invention;

[0080] Figure 2 Schematic diagram of a cross section of a microfluidic channel along the flow direction of Example 1 of the present invention;

[0081] Figure 3 It is a cross-sectional schematic diagram of the surface sensor along the flow channel direction of Example 1 of the present invention;

[0082] Figure 4 This is a schematic diagram of the processing flow of the microfluidic channel of Example 1 of the present invention;

[0083] Figure 5 This is a schematic diagram of the structure of the microfluidic channel template of Example 1 of the present invention;

[0084] Figure 6 Schematic top view of the microfluidic channel of Example 1 of the present invention;

[0085] Figure 7 This is a scanning electron microscope image of the microfluidic channel of Example 1 of the present invention;

[0086] Figure 8 Schematic diagram of the cross section of the microfluidic channel of Comparative Example 2 along the flow channel direction;

[0087] Fig. 9 Flow field simulation diagrams of the microfluidic channel of Example 1 of the present invention and the microfluidic channel of Comparative Example 2 when the flow field velocity is 10 mm / s;

[0088] Fig.10 It is a schematic diagram of the structure of the surface sensing system;

[0089] Fig.11 The reflectance spectra of the surface sensors of Example 1 and Comparative Example 1 of the present invention are as follows;

[0090] Fig.12 The antibody binding response curves of the surface sensors of Example 1 and Comparative Example 2 of the present invention are shown;

[0091] Fig.13 This is a real-time response curve diagram of the surface sensor in antigen concentration detection in Example 1 of the present invention;

[0092] Fig.14 It is a fitting curve diagram of AFP antigen concentration and displacement response in AFP antigen concentration detection;

[0093] Fig.15 This is a graph showing the change in trough displacement of the surface plasmon resonance and the change in reflectivity at a fixed wavelength of the surface sensor according to Example 1 of the present invention.

[0094] Among them, 10 is a microfluidic channel, 101 is a groove structure, 102 is a protrusion structure, 103 is a micron column, 20 is a sensor chip, 30 is a grating structure, and 40 is an auxiliary protrusion. DETAILED DESCRIPTION

[0095] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0096] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0097] Example 1

[0098] The structural schematic diagram of the microfluidic channel in Example 1 of the present invention is as follows Figure 1 shown.

[0099] The cross-sectional schematic diagram of the microfluidic channel along the flow channel direction of Example 1 of the present invention is as follows Figure 2 shown.

[0100] The cross-sectional schematic diagram of the surface sensor along the flow channel direction of Example 1 of the present invention is as follows: Figure 3 shown.

[0101] Reference Figure 2 and Figure 3 A surface sensor includes a microfluidic channel 10 and a sensor chip 20; the microfluidic channel 10 has a groove structure 101, and the groove structure is in an I-shaped shape; the bottom surface of the groove structure 101 is provided with a strip-shaped protrusion structure 102 which is perpendicular to the fluid direction and arranged in an array; micron columns 103 are distributed on the upper end surface of the protrusion structure 102, and micron columns 103 are distributed on the bottom surface of the groove structure 101 corresponding to the protrusion structures 102.

[0102] Holes with a pore size of 0.7 mm are provided at both ends of the groove structure 101 as the inlet and outlet of the detection solution; the depth of the groove structure 101 is 10 μm, and the dimension of the groove structure 101 in the width direction is 500 μm.

[0103] The height of the protruding structure 102 is 5 μm, the width of the protruding structure 102 is 30 μm, the length of the protruding structure 102 is 500 μm, and the distance between two adjacent protruding structures 102 is 20 μm.

[0104] The height of the micro-pillars 103 is 1 μm, the diameters of the micro-pillars 103 vary, ranging from 1 to 3 μm, and the micro-pillars are randomly distributed.

[0105] The difference between the sensor chip in Example 1 of the present invention and the product in Example 1 of the prior art with publication number CN 113433094A is that the metal layer in Example 1 of the present invention adopts a magnetron sputtering device to first deposit gold for 250 seconds and then deposit silver for 1000 seconds to form a gold film with a thickness of 50 nm and a silver film with a thickness of 250 nm. The rest is the same as the product in Example 1 of CN 113433094A.

[0106] The following is a preparation method of the surface sensor, which mainly involves the preparation process of the microfluidic channel template, the transfer process, and the combination process of the microfluidic channel and the sensor chip.

[0107] The schematic diagram of the processing flow of the microfluidic channel in Example 1 of the present invention is as follows Figure 4 As shown, photoresist 1 is a positive photoresist and photoresist 2 is a negative photoresist.

[0108] Reference Figure 4-6 , a method for preparing a surface sensor, comprising the following steps:

[0109] (1) Cleaning the silicon template (silicon wafer), covering the surface of the silicon template with photoresist 1AR P3740 (purchased from Alresist GmbH, Germany), exposing randomly distributed circular hole structures with a diameter of 1-3 μm on the mask material by laser through photolithography, and then etching to form circular holes with a depth of 1 μm at positions corresponding to the circular hole structures on the silicon template, thereby obtaining a silicon wafer with a circular hole structure;

[0110] (2) The surface of the silicon wafer with a circular hole structure obtained in step (1) is uniformly coated with photoresist 2AZ2035 (purchased from Merck), and photolithography is performed to expose a grating structure with a width of 20 μm in an array to form a mask material with a grating structure. The distance between two adjacent mask materials with a grating structure is 30 μm. Then, the mask material with a grating structure is used as a mask to etch the silicon wafer. The silicon wafer position corresponding to the mask material is not etched, while other areas of the silicon wafer are etched downward. The etching depth is 10 μm. After removing the mask material, a silicon wafer with an array-arranged grating structure 30 is obtained, and the grating structure 30 has the circular hole structure obtained in step (1).

[0111] (3) The surface of the silicon wafer with the grating structure 30 obtained in step (2) is covered with photoresist 2AZ2035, and photolithography is performed. The I-shaped structure is exposed by laser to form a mask material with an I-shaped structure covering the grating structure. The width of the middle position of the I-shaped structure is 500 μm and the length is 1.8 cm. The length dimension of the two ends of the I-shaped structure is 1500 μm and the width dimension is 1500 μm. The mask material with the I-shaped structure protects the covered structure to ensure that the structure will not be affected by the subsequent etching process. The silicon wafer is etched with the mask material with the I-shaped structure as a mask, and the etching depth is 10 μm to obtain a silicon wafer with auxiliary protrusions 40, that is, a microfluidic channel template is obtained, and the microfluidic channel template is arranged in an array. The structural schematic diagram of the microfluidic channel template is shown as follows: Figure 5 As shown;

[0112] (4) Transfer (reprint): Use piranha solution (concentrated H 2 SO 4 :H 2 O 2 The volume ratio is 7:3) The residual mask material on the surface of the microfluidic channel template obtained in step (3) was cleaned, rinsed with deionized water, dried with nitrogen, and baked on a hot plate for 10 minutes before use. In order to ensure the smooth separation of PDMS and the microfluidic channel template, a release agent TFOCS was evaporated on the microfluidic channel template. Specifically, 0.5 mL of the release agent was coated on the surface of the microfluidic channel template and placed in a vacuum drying oven. It was taken out after evaporation for 30 minutes; the silicone mixture (Dow Corning Sylgard184) was poured on the microfluidic channel template with the release agent evaporated, degassed in a vacuum dryer, and cured at 70°C for 2 hours, demolded, and then two holes (diameter 0.7 mm) were set at both ends of the I-shaped structure as the inlet and outlet of the detection solution, respectively, to obtain a microfluidic channel. The top view schematic diagram of the microfluidic channel is shown as follows Figure 6 As shown;

[0113] (5) In order to bond the microfluidic channel to the sensor chip to form a surface sensor, a film needs to be plated on the non-functional area of ​​the sensor chip (the area without structural features and bonded to the microfluidic channel). Because the gold metal layer of the sensor chip cannot bond to the microfluidic channel, a layer of SiO 2 As an adhesive layer, the mask covers the structural area of ​​the sensor chip, and then the sensor chip is placed in the coating equipment. A 5nm Cr / A1 layer (gold and SiO 2 No bonding, Cr / A1 is needed as an intermediate adhesion layer, magnetron sputtering, DC power supply (DC), argon (Ar) 45sccm, 100W, 30s), and then sputtering 25nm SiO 2 (Magnetron sputtering, radio frequency power (RF), argon (Ar) 60sccm, oxygen (O 2)20sccm, 150W, 1200s);

[0114] (6) The microfluidic channel and the sensor chip treated in step (5) are placed in a plasma cleaning machine and treated with 80 W oxygen plasma for 1 min. The microfluidic channel and the side of the sensor chip with the concave-convex surface structural features are then placed relative to each other, and the non-structural feature areas are fitted together to form a fluid channel, that is, a surface sensor. The microfluidic channel and the sensor chip are then heated at 70° C. for 3 min to enhance the bonding strength between the PDMS of the microfluidic channel and the sensor chip interface, thereby obtaining a surface sensor of the microfluidic channel and the sensor chip.

[0115] The scanning electron microscope image of the microfluidic channel prepared in Example 1 of the present invention is as follows: Figure 7 As shown, Figure 7 Figures (a) and (b) are SEM images at different magnifications. The protrusions of the microfluidic channel correspond to the long strip features in the SEM image, and the micrometer columns correspond to the circular features in the SEM image. Figure 7 It can be seen that the positions of the micro-pillars on and between the protrusion structures are randomly distributed, and the diameters of the micro-pillars are different.

[0116] Example 2

[0117] The only difference between Example 2 and Example 1 is that the metal layer of the sensor chip of Example 2 is deposited with gold for 1500 seconds using a magnetron sputtering apparatus to form a 300 nm gold film, and the rest is the same as Example 1.

[0118] Example 3

[0119] The only difference between Example 3 and Example 1 is that the metal layer of the sensor chip of Example 3 is formed by first depositing gold for 500 seconds and then depositing silver for 800 seconds using a magnetron sputtering apparatus to form a 100 nm gold film and a 200 nm silver film, and the rest is the same as Example 1.

[0120] Example 4

[0121] The only difference between Example 2 and Example 1 is that the metal layer of the sensor chip of Example 4 is formed by first depositing gold for 500 seconds and then depositing aluminum for 500 seconds using a magnetron sputtering apparatus to form a 100 nm gold film and a 200 nm aluminum film. The rest is the same as Example 1.

[0122] Comparative Example 1

[0123] The only difference between Comparative Example 1 and Example 1 is that the microfluidic channel of Comparative Example 1 is only provided with a protruding structure at the bottom of the groove structure without containing micro-columns, and the upper end surface of the protruding structure is not provided with micro-columns either, and the rest is the same as Example 1.

[0124] Comparative Example 2

[0125] The only difference between Comparative Example 2 and Example 1 is that the microfluidic channel of Comparative Example 2 has no protruding structure and no micro-columns, that is, the bottom surface of the groove is a flat plane, and the rest is the same as Example 1.

[0126] The cross-sectional schematic diagram of the microfluidic channel along the flow channel direction in Comparative Example 2 is as follows: Figure 8 As shown, there are no protruding structures and micro-pillars distributed on the bottom surface of the microfluidic channel, and the bottom surface of the groove is a flat plane.

[0127] Performance Testing

[0128] 1. Flow field simulation test

[0129] The static flow state of the fluid in the rectangular microfluidic channel without perturbation structure (Comparative Example 2) and the microfluidic channel with periodic groove perturbation structure (Example 1) was simulated using COMSOL software. The flow field simulation diagram of the microfluidic channel of Example 1 and Comparative Example 2 when the flow field velocity is 10 mm / s is shown in the figure below. Fig. 9 As shown, Fig. 9 Figure (a) is a flow field simulation diagram of the microfluidic channel in comparative example 2. Fig. 9 Figure (b) is a flow field simulation diagram of the microfluidic channel in Example 1; the horizontal axis Length (μm) represents the height of the microfluidic channel, and the vertical axis Length (μn) represents the length of the microfluidic channel.

[0130] Depend on Fig. 9 It can be seen that in the flat microfluidic channel of the prior art, the flow field is uniform and the flow field does not change. However, compared with the microfluidic channel in the prior art, the microfluidic channel of the present invention has a changed flow field of the fluid due to the presence of the protruding structure and the micron column, and the flow velocity increases, which can increase the efficiency of material exchange and reduce the influence of the FP interference phenomenon on the test spectrum, thereby reducing the test error and improving the detection sensitivity.

[0131] 2. Reflectance spectrum test

[0132] The reflectance spectra of the surface sensors of Example 1 and Comparative Example 1 were tested using a surface sensing system. The schematic diagram of the surface sensing system is shown in FIG. Fig.10 As shown, the surface sensors in the surface sensing system are the surface sensors of Example 1 and Comparative Example 1, respectively. The surface sensors of Example 1 and Comparative Example 1 are placed on the sample stage, and phosphate buffer solution (PBS) is passed through at a flow rate of 1 mL / h to test the reflectivity spectrum. Specifically, the light source is split into a beam through a semi-transparent and semi-reflective mirror and focused on the sample surface through a microscope objective lens to form a light spot with a diameter of 100 μm. The light spot is aligned on the sensing area ( Figure 3The yellow area in Figure 20), the light reflected from the sample surface is transmitted to the detector, and the reflectance spectrum is fed back. The hose is inserted into the inlet and outlet of the microfluidic channel, and the inlet is connected to the syringe. The syringe is installed on the syringe pump. The sample delivery flow rate is set to 1mL / h. The surface sensor is fixed on the microscope stage. The structural area of ​​the sensor chip is identified by the microscope. The system then switches to the dark field mode and starts to continuously collect the reflectance spectrum. The reflectance spectrum of the surface sensor of Example 1 and Comparative Example 1 is shown in FIG. Fig.11 As shown, the horizontal axis Wavelength represents the wavelength, and the vertical axis Reflection (%) represents the reflectivity. Fig.11 Curve a in FIG. 1 represents the reflectivity spectrum of the surface sensor of Comparative Example 1. Fig.11 Curve b in FIG. 1 represents the reflectance spectrum of the surface sensor of Example 1.

[0133] Depend on Fig.11 It can be seen that the reflectivity spectrum curve of the surface sensor in comparative example 1 has obvious fluctuations, especially the larger the wavelength, the more obvious the fluctuations, which is caused by the FP resonance between the microfluidic channel and the two planes of the sensor chip. However, due to the introduction of micron columns, the resonance between the two reflective planes of the microfluidic channel and the sensor chip is destroyed in the surface sensor of embodiment 1, and its reflectivity spectrum curve is a smooth curve without the influence of FP resonance, which can reduce the test error and improve the sensitivity.

[0134] 3. Antibody Binding Response Test

[0135] The antibody binding responsiveness of the surface sensors of Example 1 and Comparative Example 2 was tested using a surface sensing system. The specific method was as follows: the surface sensors of Example 1 and Comparative Example 2 were placed on a sample table, PBS buffer was introduced at a flow rate of 1 mL / h for 15 min, 40 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 10 mM N-hydroxysuccinimide (NHS) were introduced for activation for 15 min, PBS buffer was washed for 15 min, and then 100 μg / mL alpha-fetoprotein antibody (Anti-AFP) was introduced. The structural area of ​​the sensor chip was identified by a microscope. The surface sensing system was then switched to a dark field mode and began to continuously collect reflectance spectra. The reflectance spectrum (trough) displacement light response of the surface sensors of Example 1 and Comparative Example 2 was monitored in real time to obtain an antibody binding response curve, as shown in FIG. Fig.12 As shown. Among them, Fig.12 Figure (a) is the antibody binding response curve of the surface sensor of Comparative Example 2. Fig.12 Figure (b) is the antibody binding response curve of the surface sensor of Example 1, where the abscissa Time (min) represents time (minutes), and the ordinate Δλ (nm) represents response (nanometers).

[0136] Depend on Fig.12 It can be seen that the response curve of the surface sensor of Comparative Example 1 gradually slows down after 30 minutes, while the response curve of the surface sensor of Example 1 gradually stabilizes after 10 minutes, and the fluctuation of the red curve in Figure (a) is greater than the fluctuation of the black curve in Figure (b). This is because the perturbation structure (the protrusion structure and micron column in the microfluidic channel of Example 1) accelerates the material exchange rate in the microfluidic channel, and the introduction of the micron column structure destroys the resonance between the two reflection planes of the microfluidic channel and the sensor chip, thereby reducing the FP effect.

[0137] 4. Antigen concentration detection

[0138] The application of the surface sensor of Example 1 in antigen concentration detection specifically comprises the following steps:

[0139] (1) Sensor chip placement: The sensor chip is placed on a test bench, and phosphate buffered saline (PBS) is injected into the microfluidic channel of Example 1 at a flow rate of 1 mL / h using a syringe pump. The structural area of ​​the sensor chip is identified by a microscope, and the surface sensing system is then switched to a dark field mode, and the reflectance spectrum is continuously collected to monitor the reflectance spectrum in real time;

[0140] (2) Microfluidic channel activation: 40 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 10 mM N-hydroxysuccinimide (NHS) in 2-(N-morpholino)ethanesulfonic acid (MES) solution was injected into the microfluidic channel from the inlet for 15 min to activate the carboxyl terminal group;

[0141] (3) Antibody fixation: The activated microfluidic channel obtained in step (2) was washed with PBS. After washing for 15 min, 100 μg mL -1 Anti-AFP was fixed for 50 min to obtain the treated surface sensor;

[0142] (4) Blocking: The surface sensor treated in step (3) was washed with PBS for 15 min and then injected with 10 g L -1 bovine serum albumin (BSA), blocking the vacant space for 40 min;

[0143] (5) Testing: The surface sensor treated in step (4) was washed with PBS. After washing for 15 minutes, an AFP antigen with a concentration of 1000 ng / mL was injected for 30 minutes;

[0144] (6) Sensor chip regeneration (elution): The surface sensor treated in step (5) was washed with PBS for 15 min, and then a dissociation solution (NaH2 PO 4 / H 3 PO 4 Buffer), after 15 min of dissociation solution injection, PBS was replaced, at which time the analyte antigen was eluted;

[0145] (7) Testing: Inject 1000 ng / mL AFP antigen into the surface sensor again for 30 min;

[0146] (8) Sensor chip regeneration (elution): The surface sensor treated in step (7) was washed with PBS for 15 min, and then a dissociation solution (NaH 2 PO 4 / H 3 PO 4 Buffer), after 15 min of injection, PBS was replaced and the analyte antigen was eluted.

[0147] The above modification and testing process is monitored in real time. The AFP antigen concentration can be calculated by (5) and (7) Δλ response. The real-time response curve of the surface sensor in Example 1 in antigen concentration detection is shown in FIG. Fig.13 As shown, the abscissa Time (h) represents time (hours), and the ordinate Δλ (nm) represents response (nanometers).

[0148] Depend on Fig.13 It can be seen that after the AFP antigen test, the analyte can be dissociated by the dissociation solution, and the previously fixed antibody Anti-AFP does not fall off and remains active. After the AFP antigen is introduced again, a response can still be generated and the response curve is consistent with the previous one.

[0149] In the AFP antigen concentration detection, the AFP antigen concentration and displacement response are fitted, and the fitting curve is as follows: Fig.14 As shown, the horizontal axis C [AFP Antigen] (ng / mL) represents the AFP antigen concentration (nanograms per milliliter), the vertical axis Δλ (nm) represents the response (nanometer), and R 2 AFP =0.9943 means the correlation coefficient after fitting is 0.9943, and AFP Antigen means AFP antigen.

[0150] Depend on Fig.14 It can be seen that the surface sensor of Example 1 can detect the response of 1 ng / mL AFP antigen without the introduction of secondary antibody, and the fitting curve is in the range of 1-10000 ng mL -1 The range conforms to the S-type fitting, and the current clinical detection limit of AFP antigen is 7 ng / mL, indicating that the surface sensor prepared by the present invention has excellent sensitivity.

[0151] In addition, when performing the above data detection and analysis, the trough displacement of the surface plasmon resonance and the fixed wavelength reflectivity of the surface sensor at different time points are extracted. The changes in the trough displacement of the surface plasmon resonance and the fixed wavelength reflectivity of the surface sensor in Example 1 are as follows: Fig.15 As shown. Among them, Fig.15 Figure (a) shows the change of the trough displacement of the surface plasmon resonance, where the abscissa Time (h) represents time (hours) and the ordinate Δλ (nm) represents the response (nanometers); Fig.15 Figure (b) is a fixed wavelength reflectivity change diagram of surface plasmon resonance, where the horizontal axis Time (h) represents time (hours) and the vertical axis Reflection (%) represents reflectivity.

[0152] Depend on Fig.15 It can be seen that the change trend of the trough displacement of the surface plasmon resonance and the reflectivity at a fixed wavelength are consistent, so the analysis method can be to monitor the wavelength or to monitor the light intensity, which can provide more possibilities for sensing detection.

[0153] The surface sensors of Examples 2-4 of the present invention can also reduce the influence of FP interference on the test spectrum, reduce or even eliminate the influence of FP resonance, thereby reducing test errors and improving detection sensitivity; at the same time, they have excellent sensitivity in antigen concentration detection.

[0154] In summary, the present invention changes the flow field in the microfluidic channel by setting a protruding structure and a micron column in the microfluidic channel. Compared with the flow field without a structure, the flow velocity of the fluid increases, the material exchange rate in the microchannel is accelerated, the influence of the FP interference phenomenon on the test spectrum is reduced, and the influence of the FP resonance is reduced or even eliminated, thereby reducing the test error and improving the detection sensitivity. When the surface sensor of the present invention is used to detect AFP antigen, no secondary antibody is required, and 1 ng mL -1 The response of AFP antigen conforms to S-type fitting in the range of 1-10000 ng / mL.

[0155] The above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A surface sensor, characterized in that: It comprises a sensor chip and a microfluidic channel; the sensor chip and the microfluidic channel are placed opposite to each other with the concave and convex sides thereof to form a fluid channel; The microfluidic channel has a groove structure; the bottom surface of the groove structure is provided with a convex structure; The bottom surface of the groove structure and the upper end surface of the protrusion structure are both distributed with micrometer columns; The protrusion structures are perpendicular to the fluid direction and arranged in an array.

2. The surface sensor according to claim 1, characterized in that The depth of the groove structure is 12-33 μm; the width of the groove structure is 200-1500 μm.

3. The surface sensor according to claim 1, characterized in that: The height of the protruding structure is 4.5-10 μm; the width of the protruding structure is 18-42 μm; the length of the protruding structure is 100-1000 μm; and the distance between adjacent protruding structures is 8-32 μm.

4. The surface sensor according to claim 1, characterized in that: The diameter of the micron column is 0.9-3.3 μm; the height of the micron column is 0.8-1.2 μm.

5. The surface sensor according to claim 1, characterized in that: The sensor chip includes a substrate, a dielectric layer, and a metal layer in sequence. The dielectric layer has a plurality of dielectric grooves, and the plurality of dielectric grooves are periodically and evenly distributed. The opening width of the dielectric grooves in the direction from the groove mouth to the groove bottom gradually decreases. The metal layer is arranged on the dielectric layer and forms metal grooves corresponding to each of the dielectric grooves.

6. The method for preparing a surface sensor according to any one of claims 1 to 5, characterized in that: The following steps are involved: The microfluidic channel and the side of the sensor chip having the concave and convex surface are placed opposite to each other to obtain the surface sensor.

7. The preparation method according to claim 6, characterized in that: The method for preparing the microfluidic channel comprises the following steps: (1) forming randomly distributed micropores on the template to obtain a template having distributed micropores; (2) covering the surface of the template obtained in step (1) with a mask material, forming a mask material having a grating structure by photolithography, and etching the template using the mask material having a grating structure as a mask to form an array-arranged grating structure on the template; (3) covering the surface of the template obtained in step (2) with a mask material, forming a mask material covering the grating structure by photolithography, and etching the template using the mask material covering the grating structure as a mask to form auxiliary protrusions on the template; (4) Covering the organic silicon mixture on the template obtained in step (3), curing and separating, and obtaining a microfluidic channel.

8. The preparation method according to claim 7, characterized in that: In step (1), the diameter of the micropore is 0.9-3.3 μm; the depth of the micropore is 0.8-1.2 μm; And / or, in step (2), the height of the grating structure is 4.5-10 μm; the width of the grating structure is 8-32 μm; the length of the grating structure is 100-1000 μm; the distance between adjacent grating structures is 18-42 μm; And / or, in step (3), the height of the auxiliary protrusion is 12-33 μm; the width of the auxiliary protrusion is 200-1500 μm.

9. A surface sensor system, characterized in that: The surface sensor comprises the surface sensor according to any one of claims 1 to 5.

10. Use of the surface sensor according to any one of claims 1 to 5 or the surface sensor system according to claim 9 in detection and analysis for purposes other than disease diagnosis.

Images